Modulation of Alternaria infectoria Cell Wall Chitin and Glucan Synthesis by Cell Wall Synthase Inhibitors

Updated information and services can be found at: http://aac.asm.org/content/58/5/2894 These include: SUPPLEMENTAL MATERIAL REFERENCES

CONTENT ALERTS

Supplemental material This article cites 62 articles, 29 of which can be accessed free at: http://aac.asm.org/content/58/5/2894#ref-list-1 Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more»

Information about commercial reprint orders: http://journals.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/

Downloaded from http://aac.asm.org/ on July 5, 2014 by GEORGE MASON UNIV

Chantal Fernandes, Jorge Anjos, Louise A. Walker, Branca M. A. Silva, Luísa Cortes, Marta Mota, Carol A. Munro, Neil A. R. Gow and Teresa Gonçalves Antimicrob. Agents Chemother. 2014, 58(5):2894. DOI: 10.1128/AAC.02647-13. Published Ahead of Print 10 March 2014.

Modulation of Alternaria infectoria Cell Wall Chitin and Glucan Synthesis by Cell Wall Synthase Inhibitors Chantal Fernandes,a Jorge Anjos,a Louise A. Walker,c Branca M. A. Silva,a Luísa Cortes,a Marta Mota,a,b Carol A. Munro,c Neil A. R. Gow,c Teresa Gonçalvesa,b CNC-Center for Neurosciences and Cell Biology, University of Coimbra, Coimbra, Portugala; Faculty of Medicine, University of Coimbra, Coimbra, Portugalb; Aberdeen Fungal Group, Institute of Medical Sciences, University of Aberdeen, Aberdeen, United Kingdomc

T

he fungal cell wall has several constituents, including chitin, ␤-1,3-glucan, ␤-1,6-glucan, mannosylated mannoproteins, and phospolipomannan. The fact that this structure is essential for fungal cell growth and survival under deleterious environmental conditions and that it is absent from host cells makes it an ideal target for antifungal drugs. To date, two classes of antifungal drugs directed at fungal cell wall components have been developed, ␤-glucan synthase inhibitors, (echinocandins) and chitin synthase inhibitors (nikkomycins and polyoxins). Echinocandins are noncompetitive inhibitors of ␤-1,3-glucan synthase, and currently three drugs are approved for human antifungal therapy, caspofungin, micafungin, and anidulafungin. Nevertheless, despite the excellent safety profile and clinical efficacy of echinocandins, a number of cases of breakthrough infections have been reported. In vitro activity attenuation at higher concentrations, called the paradoxical effect, has been reported (1, 2) and is a well-established phenomenon in several fungi, such as Candida albicans and Aspergillus fumigatus (2–4). Some fungi that do not present the paradoxical effect also increase chitin levels in response to echinocandins as a salvage mechanism to compensate for decreased Fks activity (5–7). Fungi can also evade the action of echinocandins because of the presence of mutations in the FKS1 gene, which encodes the catalytic subunit of ␤-1,3-glucan synthase, that are located in two highly conserved hot-spot regions (8, 9). Several mutations have been linked to reduced susceptibility to echinocandins in Candida spp. (9, 10) and in the filamentous fungi Fusarium solani, Scedosporium prolificans, and A. fumigatus (11, 12). Also, the echinocandins increase the efficiency of phagocyte killing, as the inhibition of ␤-glucan synthase results in a pathogen that is more recognizable to host cells (13). Chitin synthases are responsible for chitin synthesis, and in fungi, these genes are grouped into seven classes (14–16). The

2894

aac.asm.org

chitin machinery is different among fungi such as C. albicans, Saccharomyces cerevisiae, and dimorphic or filamentous fungi. While S. cerevisiae and C. albicans contain three and four CHS genes, respectively, filamentous ascomycetes may contain up to 10 CHS genes (17). The multiplicity of Chs domains does not imply redundant roles in cell wall synthesis, although recent evidence suggests that C. albicans septa can be fabricated by noncanonical Chs domains (18). In this major human pathogen, Chs1 and Chs3 are responsible for canonical septum formation while Chs2 and Chs8 account for most of the in vitro chitin synthase activity and are upregulated by echinocandins (6, 19, 20). Chitin synthases belonging to classes III, V, VI, and VII are distributed only in the genomes of filamentous fungi and dimorphic yeasts that have high chitin contents in their cell walls (21). Class V and VII chitin synthases contain a myosin motor domain (MMD) in their N-terminal region (22). Functional characterization of the seven chitin synthase genes (CHS1 to CHS7) from Magnaporthe oryzae revealed that individual CHS genes play diverse roles (21). Alternaria infectoria is a filamentous fungus that contains melanin in the cell wall and belongs to the group of dematiaceous fungi (23). These are ubiquitous environmental fungi, occurring

Antimicrobial Agents and Chemotherapy

Received 9 December 2013 Returned for modification 19 January 2014 Accepted 3 March 2014 Published ahead of print 10 March 2014 Address correspondence to Teresa Gonçalves, [email protected]. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AAC.02647-13. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.02647-13

p. 2894 –2904

May 2014 Volume 58 Number 5

Downloaded from http://aac.asm.org/ on July 5, 2014 by GEORGE MASON UNIV

The present work reports the effects of caspofungin, a ␤-1,3-glucan synthase inhibitor, and nikkomycin Z, an inhibitor of chitin synthases, on two strains of Alternaria infectoria, a melanized fungus involved in opportunistic human infections and respiratory allergies. One of the strains tested, IMF006, bore phenotypic traits that conferred advantages in resisting antifungal treatment. First, the resting cell wall chitin content was higher and in response to caspofungin, the chitin level remained constant. In the other strain, IMF001, the chitin content increased upon caspofungin treatment to values similar to basal IMF006 levels. Moreover, upon caspofungin treatment, the FKS1 gene was upregulated in IMF006 and downregulated in IMF001. In addition, the resting ␤-glucan content was also different in both strains, with higher levels in IMF001 than in IMF006. However, this did not provide any advantage with respect to echinocandin resistance. We identified eight different chitin synthase genes and studied relative gene expression when the fungus was exposed to the antifungals under study. In both strains, exposure to caspofungin and nikkomycin Z led to modulation of the expression of class V and VII chitin synthase genes, suggesting its importance in the robustness of A. infectoria. The pattern of A. infectoria phagocytosis and activation of murine macrophages by spores was not affected by caspofungin. Monotherapy with nikkomycin Z and caspofungin provided only fungistatic inhibition, while a combination of both led to fungal cell lysis, revealing a strong synergistic action between the chitin synthase inhibitor and the ␤-glucan synthase inhibitor against this fungus.

A. infectoria Chitin and ␤-Glucan Synthesis

MATERIALS AND METHODS Organisms and media. Two A. infectoria clinical isolates were used in this study. One of the isolates, IMF001, was obtained by us and has been deposited at the Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Center—an institute of the Royal Netherlands Academy of Arts and Sciences as CBS 122351, and the other clinical strain (IMF006) was purchased from CBS (CBS 137.90). Strains were stored at ⫺80°C. Cultures were grown on PDA (potato dextrose agar; Difco) or YME (yeast malt extract; 4 g/liter yeast extract, 10 g/liter malt extract, 10 g/liter dextrose). Nikkomycin Z was purchased from Sigma-Aldrich, and caspofungin was a gift from Merck & Co, Inc., Rahway; NJ (material transfer agreement no. 37006). The purity of the cultures was assessed regularly by 18S rRNA gene sequencing. Preparation of inocula and growth conditions. The procedures used to prepare inocula and growth conditions were as previously described (28). Spores of A. infectoria IMF006 were collected from 2-week-old PDA plate cultures. The mycelium was submerged in liquid YME medium and scraped with an inoculation loop, and the homogenate was used as the inoculum. Strain IMF001 failed to sporulate; therefore, inocula were prepared by using fragmented hyphae. For this, fungi were grown in PDA for 2 weeks and the mycelium obtained was suspended in sterile YME medium and homogenized with glass beads (400 to 600 ␮l) in a MagNA Lyser (Roche) with mild vortexing (6,500 rpm, 1 min). The homogenate was centrifuged at 2,000 rpm to remove the larger hyphal fragments, and the supernatant containing the smaller fragments was used as the inoculum. Liquid A. infectoria IMF001 and IMF006 cultures were cultivated on YME with or without drug supplementation at 30°C with constant orbital shaking at 180 rpm for 3 days with alternating 8-h light 16-h dark cycles under an F15W T8BLB lamp. Hyphae were collected by centrifugation, washed twice in cooled water, and kept at ⫺80°C until further use. Identification of partial sequences of CHS genes. For DNA isolation, liquid cultures of A. infectoria IMF001 and IMF006 were harvested by centrifugation and DNA extractions were performed with a ZR Fungal/ Bacterial Miniprep DNA kit (Zymo Research). To identify the partial

May 2014 Volume 58 Number 5

sequences of eight fragments of CHS genes in both strains of A. infectoria, we designed several degenerate primers (see Table S1 in the supplemental material) based on the conserved regions of the Drechslera tritici-repentis and Alternaria brassicicola chitin synthases. We also used universal primers for chitin synthases. Partial fragments of CHS genes were obtained by PCR amplifications with DNA polymerase DyNA (Fisher Scientific) and 1.5 mM MgCl2 under the following conditions: 94°C for 5 min; 35 cycles of 94°C for 1 min, 48°C for 1 min (55°C for CHSG), and 72°C for 1 min; and an additional extension for 7 min at 72°C at the end of the program. The PCR products were visualized in a 1% agarose gel, and DNA fragments with the expected sizes were extracted from the gel and purified for further sequencing. Degenerate primers (OAM158, CTGAAGCTTACNATGTAYAAYGA RGAT; OAM159, CTGAAGCTTACNATGTAYAAYGARGAC) were tested to explore the possibility of the existence of further CHS genes (29). Such amplifications were unsuccessful, even at lower stringency (94°C for 3 min; 10 cycles of 92°C for 30 s, 45°C for 1 min, and 72°C for 1 min; 30 cycles of 92°C for 30 s, 40°C for 1 min, and 72°C for 1 min; and 72°C for 5 min) (30). Relative quantification of FKS1 and CHS gene expression. Total RNA isolation was performed with the MagnaPure compact RNA isolation kit (Roche). Reverse transcription (RT) of 3 ␮g of total RNA was performed with the 1st Strand cDNA synthesis kit for RT-PCR (Roche) according to the manufacturer’s instructions. The relative quantification of FKS1 and CHS gene expression was performed with the 18S rRNA gene as the reference. Real-time RT-quantitative PCRs (RT-qPCRs) were performed in a LightCycler 2.0 (Roche Diagnostics) with a LightCycler II Fast Start DNA MasterPlus SYBR green I kit (Roche Diagnostics). For the primers used in the real-time LC protocols, see Table S1 in the supplemental material. The expression level of each gene was normalized to the value of the reference (18S rRNA gene) according to the 2⫺⌬CT method, in which ⌬CT ⫽ CT,gene ⫺ CT,18S (31). Fungal morphology and viability. Fungal morphology was studied by fluorescence microscopy. The fungus was inoculated into YME with 0.1% agar supplemented with the respective drug and grown overnight at 180 rpm under alternating light and dark cycles. Before microscopic observation, 50-␮l aliquots were incubated for 2 min in the dark in a solution of 0.05% calcofluor white (CFW), 5% KOH, and 5% glycerin. Digital images were captured with an Axioskop2 Plus microscope (Carl Zeiss, Inc., Hallbergmoos, Germany) equipped with an AxioCam digital camera, a 20⫻ Plan Neofluar objective (Plan-Apochromat; Zeiss), filters (4=,6-diamidino-2-phenylindole [DAPI], fluorescein isothiocyanate), and Zeiss AxioVision image analysis capture software, and images were analyzed with ImageJ software. Images were also taken with a Zeiss LSM 510 Meta confocal microscope and viewed on a Zeiss LSM Image Browser (version 4.2.0.121; Carl Zeiss Inc.). Hyphal viability was evaluated with the FUN-1 live/dead viability kit (Molecular Probes). PDA with 0.1% agar containing the desired caspofungin and/or nikkomycin Z concentrations was inoculated with a spore suspension and incubated at 35°C for 30 h. FUN-1 was added to the growth medium at a final concentration of 10 ␮M, and the cells were incubated at 35°C for 30 min. Before the samples were placed on glass slides, CFW was added to a final concentration of 10 ␮g/ml. Cells were observed with a Zeiss Axioplan 2 microscope (Carl Zeiss, Inc.) equipped with UV-DAPI, fluorescein, and rhodamine filter sets. The images were captured with a Hamamatsu C4742-95 digital camera (Hamamatsu Photonics). Pseudocolor, contrast enhancement, and merging were applied with Openlab software, version 5 (Improvision). Cell counts were conducted in CellBIND 24-well flat-bottom microplates (Corning Inc.) at 35°C. The plates were inoculated with 103 spores/ well and left for 1 h for the spores to adhere to the bottom of each well. Unbound spores were washed away with sterile water, and 1 ml of RPMI 1640 medium with L-glutamine buffered to pH 7.0 with 0.165 M morpholinepropanesulfonic acid (MOPS) containing the desired concentration of caspofungin and/or nikkomycin Z was added to each well. Photomi-

aac.asm.org 2895

Downloaded from http://aac.asm.org/ on July 5, 2014 by GEORGE MASON UNIV

in plants, soil, food, and indoor air, that are agents of human allergies (24) and infections, causing phaeohyphomycosis— usually presenting as a subcutaneous infection (25)—that sometimes progresses to infect the central nervous system (26, 27). Here, we studied the modulatory effects of caspofungin and nikkomycin Z on the cell wall of two different strains of A. infectoria. The rationale behind the selection of these two clinical isolates from a collection of eight different isolates that did not exhibited the paradoxical effect was that one, IMF006, bears a mutation in FKS1 hot spot 2 and has a lower susceptibility to caspofungin, while the other, IMF001, has no FKS1 mutation and had a higher susceptibility to caspofungin (28). Here we demonstrate that IMF001 has a lower basal chitin cell wall level than IMF006, which, in turn, has a higher ␤-glucan content. In response to caspofungin, IMF001 increases its chitin levels to values similar to those of IMF006. The presence of caspofungin did not lead to a change in the total chitin content of strain IMF006, but the presence of nikkomycin Z led to abnormal balloon-like cells. Moreover, a synergistic action of caspofungin and nikkomycin was observed that was capable of decreasing the germination of conidia. We identified eight open reading frame (ORF) fragments from A. infectoria that had homology with fungal chitin synthase genes. Moreover, we quantified the level of CHS gene expression in both strains and concluded that the class V and VII genes of both strains are involved in the response of the fungus to caspofungin and nikkomycin. We also report that caspofungin did not markedly affect the interaction of A. infectoria conidia with macrophages.

Fernandes et al.

respective transduction to protein in A. brassicicola and D. tritici-repentis. Chitin synthase domains are represented by black boxes, while myosin motor-like domains are represented by gray boxes; boxes with dashed boundaries represent cytochrome b5-like (Cyt-b5) domains. In A. infectoria, the domain region is represented by the boxes corresponding to the nucleotide fragments that were amplified.

crographs of germinated spores were taken after 24, 48, and 72 h with an inverted microscope. Chitin and glucan content quantification. Quantification of cell wall chitin content was based on measurement of the glucosamine released by acid hydrolysis of purified cell walls (3, 32). Quantification of glucan contents was performed with the Aniline Blue assay (33), with minor changes, as described previously (28). Phagocytosis assay and cytokine quantification. The rate of A. infectoria spore phagocytosis by macrophages was studied to compare the capacities of macrophages to take up caspofungin-treated and nontreated cells. The RAW264.7 murine macrophage cell line was used (European Collection of Cell Cultures through Sigma, reference 91062702). Cells were maintained in Dulbecco’s modified Eagle medium supplemented with noninactivated fetal calf serum and sodium pyruvate at 11 mg/ml (Sigma) at 37°C in a 5% CO2 atmosphere. Phagocytosis was performed as described before (34), with slight differences. Macrophages were seeded into the wells of 96-well plates at a density of 5 ⫻ 105 cells/ml and allowed to double for 18 h at 37°C in 5% CO2. On the day of the assay, the culture medium was exchanged for fresh medium with or without caspofungin (1 ␮g/ml) and a suspension of spores (5 ⫻ 105 cells/ml) was added to each well. The final conidium-to-macrophage ratio was 1:2, and the plates were incubated at 37°C in 5% CO2 for 0, 0.5, 1.5, 3, and 6 h. After these time points, plates were kept on ice and the supernatants were collected in 1.5-ml tubes. A 200-␮l sample of fresh cold medium and 50 ␮l of 0.5% Triton X-100 (Sigma) solution was added to each well to lyse the macrophages. The cells were collected by scraping and pipetting the surface of each well and added to 1.5-ml tubes; the wells were washed once with distilled water. The tubes were then vortexed, dilutions were made, and 100-␮l suspension volumes were spread on PDA plates and incubated at 30°C with a day-night cycle for 72 h. The viability of A. infectoria conidia after macrophage ingestion was expressed as the number of CFU/ml of cell culture. For cytokine quantification, the remaining supernatants of the phagocytosis assay were centrifuged at 12,000 rpm at 4°C for 10 min, and aliquots of 80 ␮l were stored at ⫺80°C for cytokine quantification with a Bio-Plex X-Plex (Bio-Rad, Amadora, Portugal) (35). Nucleotide sequence accession numbers. The CHSA to CHSH partial nucleotide sequences determined in this study were deposited in the NCBI database under accession numbers JX436211 to JX436224, JX443517, and JX443518 (see also Table S1 in the supplemental material).

2896

aac.asm.org

RESULTS

Identification of partial CHS sequences from A. infectoria. We identified eight ORFs from A. infectoria strains IMF001 and IMF006 that have homology with other fungal chitin synthases. These partial nucleotide sequences were deposited in the NCBI database and named CHSA to CHSH (Fig. 1). Because the A. infectoria genome sequence has not been fully sequenced, we compared all of the chitin synthase gene sequences with those in the genomes of A. brassicicola (http://genome.jgi-psf.org/Altbr1/Altbr1.home.html) (36) and D. tritici-repentis (http://www.broadinstitute.org). These genomes were considered appropriate for this analysis since we showed previously that A. infectoria FKS1 (AEL31281; GenInfo Identifier [GI]: 342674147), which encodes the ␤-1,3-glucan synthase catalytic subunit, shares high nucleotide homology with the orthologues from these organisms (28). We aligned the CHS sequences and designed degenerate primers that were used to amplify nucleotide fragments from A. infectoria (see Table S1 in the supplemental material). The sequences obtained and the respective orthologues in A. brassicicola and D. tritici-repentis are represented in Fig. 1. We used the Simple Modular Architecture Research Tool software from EMBL (http: //smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL⫽1) to identify the conserved domains localized in the fragments of Chs and the corresponding proteins in A. brassicicola and D. tritici-repentis. Each chitin synthase was assigned to a class on the basis of homology with Chs orthologues from other fungi (NCBI BLAST). For the class I, II, and III Chs domains, the class assignment was unequivocal and a BLAST analysis revealed homology with several Chs domains from the same class in different fungi. ChsB and ChsC were assigned to class IV because these Chs domains do not contain an MMD domain and their Chs domains belong to Pfam03142, which is a Pfam enzyme assigned to division 2 (37). Furthermore, ChsB has a cytochrome b5-like heme/steroid binding domain and shows a high degree of homology with Fusarium fujikuroi class IV chitin synthase (GI: 517322574) (38). ChsD shows a high degree of homology with ChsE from A. fumigatus—a class V chitin synthase (2)—and class

Antimicrobial Agents and Chemotherapy

Downloaded from http://aac.asm.org/ on July 5, 2014 by GEORGE MASON UNIV

FIG 1 Representation of the amplified genomic regions of A. infectoria and comparison with the respective ORFs encoding putative chitin synthases and

A. infectoria Chitin and ␤-Glucan Synthesis

␤-glucan (B) contents of strains IMF001 and IMF006 are shown. The strains were cultivated in liquid cultures for 3 days with alternating 8-h light and 16-h dark periods on YME with or without 1 ␮g/ml caspofungin and/or 5 ␮g/ml nikkomycin Z. Results are the mean ⫾ the standard error of the mean of triplicates of three independent experiments (Student’s t test). *, P ⬍ 0.05; **, P ⬍ 0.03; ***, P ⬍ 0.01.

Va (35). Curiously, A. brassicicola class V sequence AB05905.1 seems to be truncated compared to its orthologue PTRG_05004.1, and its myosin motor-like domains and N termini are shortened (Fig. 1). ChsE shows a high degree of homology with ChsVb from Fusarium oxysporum—a class VII chitin synthase (39)—and class Vb A. fumigatus chitin synthase (37). ChsF from A. brassicicola and D. tritici-repentis were classified as putative class VI enzymes because the Chs domain was recognized by NCBI as Pfam03142, but these enzymes also show a high degree of homology with the class VI Chs domains of other fungi. Effects of caspofungin and nikkomycin Z on cell wall chitin and glucan contents. The cell wall chitin and ␤-glucan contents of mycelia of the two strains after exposure to cell wall-acting antibiotics were quantified. As shown in Fig. 2A, the basal level of chitin in the cell wall of strain IMF006 was almost twice that of strain IMF001. Inhibition of ␤-1,3-glucan synthase by treatment with 1 ␮g/ml caspofungin for 3 days led to a compensatory increase in chitin in strain IMF001, while the levels of chitin in strain IMF006 remained almost unchanged upon caspofungin treatment. In contrast, treatment with nikkomycin Z led to a significant increase in the chitin contents of both strains but this was more pronounced in strain IMF006 than in strain IMF001 (Fig. 2A). We therefore conclude that only some of the chitin synthases of A. infectoria are inhibited by nikkomycin Z and that other chitin synthases account for the observed elevation of chitin content by nikkomycin Z.

May 2014 Volume 58 Number 5

aac.asm.org 2897

Downloaded from http://aac.asm.org/ on July 5, 2014 by GEORGE MASON UNIV

FIG 2 Modulation of cell wall components by antifungals. Chitin (A) and

␤-Glucan quantification revealed that IMF001 had a higher basal level of ␤-glucan than IMF006 in control cultures. Nevertheless, a decrease in ␤-glucan content occurred in both strains upon treatment with caspofungin (Fig. 2B). The ␤-glucan content did not change when the strains were exposed to nikkomycin Z (Fig. 2B), a result that was predicted since this drug did not affect the cell wall chitin content (Fig. 2A). When A. infectoria strains were exposed to a combination of caspofungin and nikkomycin Z, strain IMF006 increased its chitin content to the highest levels and the glucan level was similar to that determined in the presence of caspofungin alone. In strain IMF001, the chitin content increased above the basal level, to a value similar to that observed upon exposure to caspofungin alone (Fig. 2A and B). Fungal morphology changes. As shown above, strains IMF001 and IMF006 have different susceptibilities to cell wall-acting antifungals. Morphological differences were also observed in these strains in the absence of any antifungal treatment (Fig. 3). For example, strain IMF006 formed abundant conidia but spores were scarce in strain IMF001. For this reason, assays involving spores could be done only with strain IMF006. The morphology of strain IMF001 was unchanged in the presence of nikkomycin Z or caspofungin. The accumulation of chitin, visualized by CFW fluorescence, was uniform along the hyphal trunk under all of the conditions tested. However, strain IMF006, when exposed to nikkomycin Z, exhibited a ballooning phenotype that did not occur when it was grown in the presence of caspofungin (Fig. 3). When IMF006 was grown with or without caspofungin, we observed by confocal microscopy (Fig. 4) that most of the new chitin occurred within balloon-like structures (Fig. 4). Greater accumulation of chitin in the septal rings was also evident (Fig. 4A, B, and E, confocal z-stack). This indicates that the biosynthesis of chitin during nikkomycin Z exposure involves preferential deposition at septa rather than in the hyphal lateral cell wall (Fig. 4). The higher plasticity of strain IMF006 morphology, together with the previously reported lower caspofungin susceptibility of this strain than other A. infectoria strains (28), led us to further examine the morphological changes that occur during spore germination. In the presence of caspofungin, significant structural alterations were observed during hyphal development from the onset of spore germination (Fig. 5B), where short, stubby, highly branched hyphae were observed. These effects are similar to those described in A. fumigatus (40), except that no cell lysis was detected at the hyphal tips. Nikkomycin Z also induced structural abnormalities during A. infectoria spore germination and in hyphae (Fig. 5C), but these were more localized, with occasional swollen, balloon-like cells observed in what appeared to be otherwise normal hyphae. The frequency of cell swelling was higher near the germinated spore and decreased as hyphal growth progressed, suggesting the existence of an adaptive mechanism that might progressively increase drug tolerance upon hyphal growth. CFW staining revealed that the abnormal cells exhibited higher fluorescence intensity, reflecting the increase in chitin content, upon nikkomycin Z treatment. The observed effects of nikkomycin Z were similar over the range of concentrations tested (0.5 to 32 ␮g/ml). Even at the highest concentration tested, neither caspofungin (16 ␮g/ml) nor nikkomycin Z (32 ␮g/ml) alone was capable of completely blocking hyphal growth or causing cell lysis in A. infectoria IMF006 (see Fig. S2 in the supplemental material).

Fernandes et al.

were stained with CFW. Images were acquired with an Observer Z1 microscope (Carl Zeiss, Jena, Germany), with a Plan-Apochromat 20⫻ (numerical aperture 0.8) objective.

However, the combination of caspofungin and nikkomycin Z completely blocked hyphal growth in A. infectoria IMF006 and restricted vestigial growth to the production of clusters of rounded cells (Fig. 5D) that were unable to germinate or form hyphae. Moreover, in the CFW- and FUN1-costaining images, it

was possible to visualize a significant number of dead and lysed cells that were absent when the fungal cells were incubated with the individual antifungals. Live imaging revealed that following the germination process in the presence of caspofungin (2 ␮g/ml) and nikkomycin Z (2 ␮g/ml), IMF006 spores germinated into

FIG 4 Changes in morphology and chitin distribution upon exposure to nikkomycin Z. Shown is a three-dimensional projection obtained by confocal imaging

of chitin stained with CFW in strain IMF006 grown in YME without (A, C) or with (B, D) 5 ␮g/ml nikkomycin Z. For each condition, maximum-intensity projections of z-stack images were obtained with FIJI software and the mean fluorescence intensity at the septal rings (arrows) was quantified (E). Cell imaging was performed with a Zeiss LSM 510 Meta confocal microscope with a Plan-Apochromat 63⫻ (numerical aperture 1.4) oil objective.

2898

aac.asm.org

Antimicrobial Agents and Chemotherapy

Downloaded from http://aac.asm.org/ on July 5, 2014 by GEORGE MASON UNIV

FIG 3 Fungal morphology of A. infectoria strains IMF001 and IMF006 grown in YME with or without 1 ␮g/ml caspofungin or 5 ␮g/ml nikkomycin Z. Cell walls

A. infectoria Chitin and ␤-Glucan Synthesis

round, balloon-like spores that, in most cases, eventually burst (see Video S3 in the supplemental material). To quantify the combined effect of both drugs, A. infectoria IMF006 spores were attached to the bottom of microplate wells and spore germination and hyphal growth were monitored daily for 3 days. Exposure to a single antifungal (caspofungin or nikkomycin Z) did not prevent spore germination or hyphal growth (Fig. 6A). However, A. infectoria pregrown hyphae displayed the structural abnormalities described above after 3 days of incubation and the fungal mat at the bottom of the microplate wells was almost as dense as that of untreated controls (Fig. 6B). The addition of 2 ␮g/ml caspofungin plus 2 ␮g/ml nikkomycin Z to the medium not only prevented spore germination but also resulted in the complete absence of any visible growth after the first day in 12% of the spores. The percentage of spores with arrested growth after the first day increased to 37% when the nikkomycin Z concentration was increased to 16 ␮g/ml.

May 2014 Volume 58 Number 5

Quantification of CHS and FKS gene expression. We next determined the differential effect of caspofungin on the transcriptional regulation of the eight putative chitin synthase genes and of the FKS1 gene by real-time RT-qPCR. Overall, the levels of gene expression (both CHS and FKS1) in strain IMF001 were higher than the levels of expression in strain IMF006 (Fig. 7). The fragment encoding CHSC exhibited low expression levels under all of the conditions tested, preventing accurate assessment of its expression levels by real-time RT-qPCR. The inhibition of glucan synthesis by caspofungin resulted in a 3-fold increase in CHSD expression in IMF001 and a 4-fold increase in IMF006 compared to that in untreated controls. The CHS gene with the highest expression level in strain IMF001 was CHSE (class VII), and its expression was further increased by caspofungin treatment (Fig. 7A). It was concluded that the compensatory mechanism of increasing chitin synthesis in the presence of the ␤-glucan inhibitor in IMF001 was due

aac.asm.org 2899

Downloaded from http://aac.asm.org/ on July 5, 2014 by GEORGE MASON UNIV

FIG 5 Effects of caspofungin and nikkomycin Z on the hyphal structure of A. infectoria IMF006. A drug-free control (A), treatment with caspofungin (2 ␮g/ml) (B), treatment with nikkomycin Z (2 ␮g/ml) (C), and treatment with caspofungin (2 ␮g/ml) and nikkomycin Z (2 ␮g/ml) (D) are shown. Scale bars, 65 ␮m in panels A, B, and C and 15 ␮m in panel D. CFW was used to visualized chitin accumulation along the hyphae. Hyphal viability was evaluated with the FUN-1 live/dead viability kit with a Zeiss Axioplan 2 microscope (Carl Zeiss, Inc.) equipped with UV-DAPI, fluorescein, and rhodamine filter sets. The images were captured with a Hamamatsu C4742-95 digital camera (Hamamatsu Photonics).

Fernandes et al.

(B), treatment with nikkomycin Z (NZ; 2 ␮g/ml) (C), treatment with caspofungin (2 ␮g/ml) and nikkomycin Z (2 ␮g/ml) (D), and quantification of fungal growth after 72 h are shown. White, normal hyphae; gray, abnormal hyphae; black, no hyphae formed. The images in panels A to D were taken after 72 h of incubation in RPMI 1640 medium at 30°C. Hyphal growth was evaluated by comparing photomicrographs of germinated spores taken after 24, 48, and 72 h in an inverted microscope.

primarily to the overexpression of CHSE and CHSD (class V). As shown in Fig. 2, this compensatory mechanism did not occur in IMF006, probably because this strain has a high basal level of chitin. Moreover, in strain IMF006, no further increase

in chitin content occurred at concentrations of caspofungin higher than 1 ␮g/ml (data not shown). FKS1 was upregulated in IMF006 in response to caspofungin, while in IMF001 it was downregulated (28). Since IMF001 contains more ␤-glucan

FIG 7 A. infectoria CHS and FKS gene expression. A. infectoria was incubated in the absence (control condition) (⫺) or presence (⫹) of 1 ␮g/ml caspofungin

(A) or 5 ␮g/ml nikkomycin Z (B). Relative gene expression was determined by RT-qPCR with the 18S rRNA gene as the reference. The method used to analyze data from real-time assays and to express CHS and FKS gene expression levels for each condition in relation to the 18S rRNA gene was the 2⫺⌬CT method, in which ⌬CT ⫽ CT,gene ⫺ CT,18S (31). The results are presented as the expression of each gene by IMF001 and IMF006 under control conditions and with the antifungal. Results are the average value ⫾ the standard error of the mean of at least duplicates of two independent experiments. Statistical analyses were performed with GraphPad Prism software and the two-sample Student t test (*, P ⬍ 0.05; **, P ⬍ 0.03; ***, P ⬍ 0.01).

2900

aac.asm.org

Antimicrobial Agents and Chemotherapy

Downloaded from http://aac.asm.org/ on July 5, 2014 by GEORGE MASON UNIV

FIG 6 Combined effect of caspofungin and nikkomycin Z on A. infectoria IMF006 growth. A drug-free control (A), treatment with caspofungin (CAS; 2 ␮g/ml)

A. infectoria Chitin and ␤-Glucan Synthesis

(M␾ ⫹ conidia) or presence of 1 ␮g/ml caspofungin (M␾ ⫹ conidia ⫹ Caspo). At the time points indicated, samples were taken for assessment of conidial viability, which was quantified with a CFU assay (A) and the supernatants were collected to quantify cytokines (B) as described in Materials and Methods. (A) The results are expressed as numbers of CFU/ml of cell culture (white columns, macrophages plus conidia; black columns, macrophages plus conidia plus caspofungin). (B) Cytokines were quantified with a Bio-Plex X-Plex (Bio-Rad). IL-10, interleukin-10; GM-CSF, granulocyte-macrophage colony-stimulating factor; TNF-␣, tumor necrosis factor alpha; MIP-1␣, macrophage inflammatory protein 1 alpha; INF-␥, gamma interferon.

than IMF006, FKS1 upregulation in IMF006 might account for its lower susceptibility to caspofungin. In the presence of 5 ␮g/ml nikkomycin Z, the genes CHSA (class II), CHSD, and CHSG (class I) were upregulated in both strains. In strain IMF006, CHSE gene expression was also significantly increased (Fig. 7B). Upregulation of these genes is likely to account for the increase in cell wall chitin in both strains (Fig. 2). Macrophage response to A. infectoria in the presence of caspofungin. In C. albicans (41, 42) and in A. fumigatus (43), it was shown that exposure to caspofungin leads to a disturbance of the cell wall that alters the rates of internalization and activation of phagocytic cells. We cocultured a macrophage cell line with A. infectoria conidia (strain IMF006) to evaluate how

May 2014 Volume 58 Number 5

phagocytosis is affected by the presence of caspofungin. We measured the internalization of conidia by RAW264.7 macrophages, the viability of the fungal cells inside the macrophage, and the differential production of cytokines in the absence or presence of 1 ␮g/ml caspofungin. Macrophages internalized most of the conidia during the first half hour (Fig. 8A). The viability of conidia inside macrophages was unchanged for the first 6 h of exposure to macrophages, and this was not affected by caspofungin exposure (P ⫽ 0.6459). Moreover, the release of proinflammatory and anti-inflammatory cytokines by macrophages was not affected by caspofungin treatment (Fig. 8B), even at stages when the conidia had begun to germinate (results not shown). Changes in the immune recognition of mature hyphae were not examined in this analysis.

aac.asm.org 2901

Downloaded from http://aac.asm.org/ on July 5, 2014 by GEORGE MASON UNIV

FIG 8 Phagocytosis of A. infectoria conidia by RAW264.7 cells and cytokine production. RAW264.7 macrophage cells were infected with conidia in the absence

Fernandes et al.

DISCUSSION

2902

aac.asm.org

enzymes may be able to compensate for the inhibition of others. Differential effects on chitin synthase isozymes have been reported previously to influence chitin synthase inhibitor efficacy. Gaughran and collaborators demonstrated that nikkomycin Z is a selective inhibitor of chitin synthases 1 and 3 in S. cerevisiae and proved the antifungal resistance of chitin synthase 2 in vitro (48). The redundancy within CHS multigene families may be an important factor in limiting the in vivo efficacy of nikkomycin Z and other compounds that affect in vitro chitin synthase (48, 49). In A. fumigatus, it was reported that chitin cell wall content was not affected by nikkomycin Z treatment but was markedly affected by a combination of caspofungin and nikkomycin Z (50, 51). Also, in this strain, the combination of the two antifungals led to changes in cell wall chitin and ␤-glucan contents. The synergy between nikkomycin Z and caspofungin suggests that this might be exploitable in antifungal chemotherapy strategies. This synergy has also been described in vitro for other human pathogens (7, 50, 52, 53) but until now, this has not been thoroughly tested in vivo. Recently, it was proved that nikkomycin Z is efficient in the eradication of naturally occurring respiratory coccidioidomycosis in dogs (54). Here we report that A. infectoria possesses at least eight putative chitin synthase genes. This multiplicity of isoforms implies specificity of function, as it occurs in other fungi (21, 55). We found that the expression of some CHS genes was altered, depending on the antifungal and on the A. infectoria strain. In another melanized human pathogen, Wangiella (Exophiala) dermatitidis, evidence of transcriptional compensation in various chs mutant backgrounds has been found (56). Compensatory expression of one CHS gene in response to defects in other CHS genes has also been reported in C. albicans (3). This compensatory expression change occurs in response to decreased glucan levels and the formation of salvage septa (4, 6, 18). We show here that CFW fluorescence was higher in hyphal septa of caspofungin-treated cells. Also, the abnormal balloon-like cells exhibited higher fluorescence intensity, indicating that the increase in chitin content measured upon nikkomycin Z treatment might be a localized effect that occurs only at points of local cell wall stress. The molecular mechanism of protection of A. infectoria against caspofungin was different in strains IMF001 and IMF006. In IMF006, only the expression of the class V chitin synthase gene CHSD was significantly increased, suggesting that the enzyme encoded by this gene may be required for protection against ␤-glucan damage. The importance of class V chitin synthases, which contain an MMD, is well documented for numerous fungi. In W. dermatitidis, the product of CHS5 is required for the sustained growth of this organism at 37°C and for virulence (57). In the plant pathogen Ustilago maydis, a class V myosin is required for the initial steps of plant infection (58) and both Chs and MMD are required for virulence (59). In Fusarium oxysporum, ChsV is required for pathogenesis and mediates resistance to plant defense compounds (47). Also, A. nidulans ⌬csma mutants display abnormalities in hyphal morphology, including the formation of balloons and intrahyphal hyphae, as well as hyphal lysis, particularly under low osmotic conditions (60). In the present study, in both of the A. infectoria strains tested, CHSE, a class VII Chs that contain an MMD, was overexpressed in response to caspofungin and nikkomycin Z. Class VII enzymes have also been demonstrated to be essential for virulence in several plant pathogens (21, 61) and,

Antimicrobial Agents and Chemotherapy

Downloaded from http://aac.asm.org/ on July 5, 2014 by GEORGE MASON UNIV

Fungi of the genus Alternaria are opportunistic pathogens with preeminent importance in human health in relation to allergies, particularly severe asthma (44, 45). Regardless of the growing interest in these fungi, knowledge about the molecular mechanisms of pathogenesis and susceptibility to antifungals has not advanced as it has for the major human fungal pathogens. Previously, we characterized the A. infectoria genes that encode the ␤-1,3-glucan synthase, FKS1, and its regulatory subunit, RHO. Hot spot 2 mutations were recognized in the FKS1 gene of one of the strains, IMF006, and none of the eight strains tested exhibited the paradoxical effect, (28). In the present work we compared the IMF006 strain with another clinical isolate of A. infectoria, IMF001, that had no hot spot 2 mutation and high susceptibility to caspofungin, in terms of its response to caspofungin and nikkomycin Z. We show that A. infectoria is endowed with at least eight genes that code for chitin synthases and that strain IMF006 had a high basal chitin content and did not activate the salvage mechanism in the presence of caspofungin. In contrast, strain IMF001 had a lower basal cell wall chitin and, regardless of its ␤-glucan content, activated the cell wall salvage mechanism in the presence of caspofungin, leading to chitin content elevation to a level that approximated the basal chitin level in IMF006. Therefore, IMF006 is endowed with a cell wall more prepared to cope with antifungals that affect fungal cell wall integrity because of (i) an FKS1 hot spot 2 mutation and (ii) a cell wall with high basal chitin content. These two biological properties may account for its lower susceptibility to caspofungin (28). We conclude that the robustness of the fungal cell wall of A. infectoria is dependent in part on its chitin content. This supports the observation that C. albicans cells with a high chitin content also have reduced echinocandin susceptibility both in vivo and in vitro (5). Caspofungin exposure increases the inflammatory response of macrophages to C. albicans because of exposure of ␤-glucan (41, 42, 46). Also, in A. fumigatus, caspofungin treatment results in a decrease in the release of tumor necrosis factor alpha during conidial interaction with macrophages but increases the inflammatory response to hyphae (43). These changes were related to differential ␤-glucan exposure at the surface of the fungal cells. Here we found that the internalization of A. infectoria conidia by macrophages was not caspofungin sensitive. Even after spore germination, little change in the viability and in the release of cytokines was observed. These results might be interpreted as being due to the fact that the germinating spore is protected from caspofungin inside the macrophage. We also observed that growth in nikkomycin Z could induce hyphal ballooning, but only in strain IMF006 (Fig. 3 and 4). In the presence of both nikkomycin Z and caspofungin, the spores of this strain failed to germinate and instead became highly swollen and had decreased cell viability (Fig. 5 and 6; see Fig. S2 and Video S3 in the supplemental material). This phenotype was also described in several CHS mutants, such as a C. albicans chs1⌬ mutant (20); Fusarium oxysporum ⌬chsV and ⌬chsVb mutants and a double ⌬chsV ⌬chsVb mutant (39, 47); and a Colletotrichum graminicola ⌬ChsV mutant (17). Another striking observation was that the chitin content increased when strains were grown in the presence of nikkomycin Z. This apparent paradox is explained by the facts that this inhibitor does not inhibit all chitin synthase isoforms and residual active

A. infectoria Chitin and ␤-Glucan Synthesis

together with class V Chs, seem to play a crucial structural role in the human pathogen A. fumigatus and in A. nidulans (62, 63). Overall, this work reinforces the observation that fungi can modulate their cell wall composition to abrogate antifungal exposure, and in some cases, this eventually may lead to breakthrough infections. A strong synergistic effect when ␤-glucan and chitin synthesis inhibitors are combined suggests novel therapeutic approaches to the treatment of opportunistic infections with dematiaceous and other fungi. ACKNOWLEDGMENTS

REFERENCES 1. Wiederhold NP. 2007. Attenuation of echinocandin activity at elevated concentrations: a review of the paradoxical effect. Curr. Opin. Infect. Dis. 20:574 –578. http://dx.doi.org/10.1097/QCO.0b013e3282f1be7f. 2. Lenardon MD, Munro CA, Gow NAR. 2010. Chitin synthesis and fungal pathogenesis. Curr. Opin. Microbiol. 13:416 – 423. http://dx.doi.org/10 .1016/j.mib.2010.05.002. 3. Munro CA, Selvaggini S, de Bruijn I, Walker L, Lenardon MD, Gerssen B, Milne S, Brown AJ, Gow NAR. 2007. The PKC, HOG and Ca2⫹ signalling pathways co-ordinately regulate chitin synthesis in Candida albicans. Mol. Microbiol. 63:1399 –1413. http://dx.doi.org/10.1111/j.1365 -2958.2007.05588.x. 4. Fortwendel JR, Juvvadi PR, Perfect BZ, Rogg LE, Perfect JR, Steinbach WJ. 2010. Transcriptional regulation of chitin synthases by calcineurin controls paradoxical growth of Aspergillus fumigatus in response to caspofungin. Antimicrob. Agents Chemother. 54:1555–1563. http://dx.doi.org /10.1128/AAC.00854-09. 5. Lee KK, MacCallum DM, Jacobsen MD, Walker LA, Odds FC, Gow NAR, Munro CA. 2012. Elevated cell wall chitin in Candida albicans confers echinocandin resistance in vivo. Antimicrob. Agents Chemother. 56:208 –217. http://dx.doi.org/10.1128/AAC.00683-11. 6. Walker LA, Munro CA, de Bruijn I, Lenardon MD, McKinnon A, Gow NAR. 2008. Stimulation of chitin synthesis rescues Candida albicans from echinocandins. PLoS Pathog. 4:e1000040. http://dx.doi.org/10.1371 /journal.ppat.1000040. 7. Walker LA, Gow NAR, Munro CA. 2013. Elevated chitin content reduces the susceptibility of Candida species to caspofungin. Antimicrob. Agents Chemother. 57:146 –154. http://dx.doi.org/10.1128/AAC.01486-12. 8. Park S, Kelly R, Kahn JN, Robles J, Hsu MJ, Register E, Li W, Vyas V, Fan H, Abruzzo G, Flattery A, Gill C, Chrebet G, Parent SA, Kurtz M, Teppler H, Douglas CM, Perlin DS. 2005. Specific substitutions in the echinocandin target Fks1p account for reduced susceptibility of rare laboratory and clinical Candida sp. isolates. Antimicrob. Agents Chemother. 49:3264 –3273. http://dx.doi.org/10.1128/AAC.49.8.3264-3273.2005. 9. Perlin DS. 2007. Resistance to echinocandin-class antifungal drugs. Drug Resist. Updat. 10:121–130. http://dx.doi.org/10.1016/j.drup.2007.04.002. 10. Garcia-Effron G, Park S, Perlin DS. 2009. Correlating echinocandin MIC and kinetic inhibition of fks1 mutant glucan synthases for Candida albicans: implications for interpretive breakpoints. Antimicrob. Agents Chemother. 53:112–122. http://dx.doi.org/10.1128/AAC.01162-08. 11. Katiyar SK, Edlind TD. 2009. Role for Fks1 in the intrinsic echinocandin resistance of Fusarium solani as evidenced by hybrid expression in Saccharomyces cerevisiae. Antimicrob. Agents Chemother. 53:1772–1778. http: //dx.doi.org/10.1128/AAC.00020-09.

May 2014 Volume 58 Number 5

aac.asm.org 2903

Downloaded from http://aac.asm.org/ on July 5, 2014 by GEORGE MASON UNIV

This study was partly supported by a Merck, Sharp & Dohme, Inc., medical school grant (P-1599) and by a project funded by FCT-Fundação para a Ciência e Tecnologia (PTDC/SAU-ESA/108636/2008, cofunded by COMPETE and FEDER, and PEst-C/SAU/LA0001/2013-2014). J.A. was the recipient of a postdoctoral fellowship from FCT-Fundação para a Ciência e Tecnologia (SFRH/BPD/34419/2006). C.F. is a recipient of a postdoctoral fellowship from FCT-Fundação para a Ciência e Tecnologia (SFRH/BPD/63733/2009). B.M.A.S. was a recipient of a research fellowship within the scope of FCT project PTDC/SAU-ESA/108636/2008. N.A.R.G. is supported by the Wellcome Trust (080088, 086827, 075470, and 097377). We acknowledge Frank Odds and N. Empadinhas for helpful and stimulating discussions.

12. Howard SJ, Arendrup MC. 2011. Acquired antifungal drug resistance in Aspergillus fumigatus: epidemiology and detection. Med. Mycol. 49(Suppl 1):S90 –S95. http://dx.doi.org/10.3109/13693786.2010.508469. 13. Mora-Montes HM, Netea MG, Ferwerda G, Lenardon MD, Brown GD, Mistry AR, Kullberg B-J, O’Callaghan CA, Sheth CC, Odds FC, Brown AJP, Munro CA, Gow NAR. 2011. Recognition and blocking of innate immunity cells by Candida albicans chitin. Infect. Immun. 79:1961–1970. http://dx.doi.org/10.1128/IAI.01282-10. 14. Choquer M, Boccara M, Goncalves IR, Soulie MC, Vidal-Cros A. 2004. Survey of the Botrytis cinerea chitin synthase multigenic family through the analysis of six Euascomycetes genomes. Eur. J. Biochem. 271:2153– 2164. http://dx.doi.org/10.1111/j.1432-1033.2004.04135.x. 15. Chigira Y, Abe K, Gomi K, Nakajima T. 2002. chsZ, a gene for a novel class of chitin synthase from Aspergillus oryzae. Curr. Genet. 41:261–267. http://dx.doi.org/10.1007/s00294-002-0305-z. 16. Roncero C. 2002. The genetic complexity of chitin synthesis in fungi. Curr. Genet. 41:367–378. http://dx.doi.org/10.1007/s00294-002-0318-7. 17. Werner S, Sugui JA, Steinberg G, Deising HB. 2007. A chitin synthase with a myosin-like motor domain is essential for hyphal growth, appressorium differentiation, and pathogenicity of the maize anthracnose fungus Colletotrichum graminicola. Mol. Plant Microbe Interact. 20:1555– 1567. http://dx.doi.org/10.1094/MPMI-20-12-1555. 18. Walker L, Lenardon MD, Munro CA, Gow NAR. 2013. Cell wall stress induces alternative fungal cytokinesis and septation strategies. J. Cell Sci. 126:2668 –2677. http://dx.doi.org/10.1242/jcs.118885. 19. Gow NAR. 1994. Growth and guidance of the fungal hypha. Microbiology 140:3193–3205. http://dx.doi.org/10.1099/13500872-140-12-3193. 20. Munro CA, Whitton RK, Hughes B, Rella M, Selvaggini S, Gow NAR. 2003. CHS8 the fourth chitin synthase gene of Candida albicans contributes to in vitro chitin synthase activity but is dispensable for growth. Fungal Genet. Biol. 40:146 –158. http://dx.doi.org/10.1016/S1087-1845(03) 00083-5. 21. Kong LA, Yang J, Li GT, Qi LL, Zhang YJ, Wang CF, Zhao WS, Xu JR, Peng YL. 2012. Different chitin synthase genes are required for various developmental and plant infection processes in the rice blast fungus Magnaporthe oryzae. PLoS Pathog. 8:e1002526. http://dx.doi.org/10.1371 /journal.ppat.1002526. 22. Takeshita N, Yamashita S, Ohta A, Horiuchi H. 2006. Aspergillus nidulans class V and VI chitin synthases CsmA and CsmB, each with a myosin motor-like domain, perform compensatory functions that are essential for hyphal tip growth. Mol. Microbiol. 59:1380 –1394. http://dx.doi.org/10 .1111/j.1365-2958.2006.05030.x. 23. de Hoog GS, Guarro J, Gené J, Figueras MJ. 2000. Atlas of clinical fungi, 2nd ed. Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands. 24. Knutsen AP, Bush RK, Demain JG, Denning DW, Dixit A, Fairs A, Greenberger PA, Kariuki B, Kita H, Kurup VP, Moss RB, Niven RM, Pashley CH, Slavin RG, Vijay HM, Wardlaw AJ. 2012. Fungi and allergic lower respiratory tract diseases. J. Allergy Clin. Immunol. 129:280 –291. http://dx.doi.org/10.1016/j.jaci.2011.12.970. 25. Gilaberte M, Bartralot R, Torres JM, Reus FS, Rodríguez V, Alomar A, Pujol RM. 2005. Cutaneous alternariosis in transplant recipients: clinicopathologic review of 9 cases. J. Am. Acad. Dermatol. 52:653– 659. http://dx .doi.org/10.1016/j.jaad.2004.10.875. 26. Li DM, de Hoog GS. 2009. Cerebral phaeohyphomycosis—a cure at what lengths? Lancet Infect. Dis. 9:376 –383. http://dx.doi.org/10.1016/S1473 -3099(09)70131-8. 27. Hipólito E, Faria E, Alves AF, de Hoog GS, Anjos J, Gonçalves T, Morais PV, Estevão H. 2009. Alternaria infectoria brain abscess in a child with chronic granulomatous disease. Eur. J. Clin. Microbiol. Infect. Dis. 28:377–380. http://dx.doi.org/10.1007/s10096-008-0623-2. 28. Anjos J, Fernandes C, Silva BM, Quintas C, Abrunheiro A, Gow NAR, Gonçalves T. 2012. ␤(1,3)-Glucan synthase complex from Alternaria infectoria, a rare dematiaceous human pathogen. Med. Mycol. 50:716 –725. http://dx.doi.org/10.3109/13693786.2012.675525. 29. Mandel MA, Galgiani JN, Kroken S, Orbach MJ. 2006. Coccidioides posadasii contains single chitin synthase genes corresponding to classes I to VII. Fung. Genet. Biol. 43:775–788. http://dx.doi.org/10.1016/j.fgb .2006.05.005. 30. Caballero OL, Villa LL, Simpson AJG. 1995. Low stringency-PCR (LSPCR) allows entirely internally standardized DNA quantitation. Nucleic Acids Res. 23:192–193. http://dx.doi.org/10.1093/nar/23.1.192. 31. Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data

Fernandes et al.

32.

33.

34.

36.

37. 38.

39.

40.

41.

42.

43.

44. 45.

46.

2904

aac.asm.org

47.

48. 49. 50.

51.

52.

53.

54. 55.

56.

57.

58.

59.

60.

61. 62.

63.

fungi from the immune system. PLoS Pathog. 2:e35. http://dx.doi.org/10 .1371/journal.ppat.0020035. Madrid MP, Di Pietro A, Roncero MI. 2003. Class V chitin synthase determines pathogenesis in the vascular wilt fungus Fusarium oxysporum and mediates resistance to plant defence compounds. Mol. Microbiol. 47:257–266. http://dx.doi.org/10.1046/j.1365-2958.2003.03299.x. Gaughran JP, Lai MH, Kirsch DR, Silverman SJ. 1994. Nikkomycin Z is a specific inhibitor of Saccharomyces cerevisiae chitin synthase isozyme Chs3 in vitro and in vivo. J. Bacteriol. 176:5857–5860. Naider F, Becker JM. 1988. Peptide transport in Candida albicans: implications for the development of antifungal agents. Curr. Top. Med. Mycol. 2:170 –198. http://dx.doi.org/10.1007/978-1-4612-3730-3_5. Verwer PE, van Duijn ML, Tavakol M, Bakker-Woudenberg IA, van de Sande WW. 2012. Reshuffling of Aspergillus fumigatus cell wall components chitin and ␤-glucan under the influence of caspofungin or nikkomycin Z alone or in combination. Antimicrob. Agents Chemother. 56: 1595–1598. http://dx.doi.org/10.1128/AAC.05323-11. Chiou CC, Mavrogiorgos N, Tillem E, Hector R, Walsh TJ. 2001. Synergy, pharmacodynamics, and time-sequenced ultrastructural changes of the interaction between nikkomycin Z and the echinocandin FK463 against Aspergillus fumigatus. Antimicrob. Agents Chemother. 45: 3310 –3321. http://dx.doi.org/10.1128/AAC.45.12.3310-3321.2001. Sandovsky-Losica H, Shwartzman R, Lahat Y, Segal E. 2008. Antifungal activity against Candida albicans of nikkomycin Z in combination with caspofungin, voriconazole or amphotericin B. J. Antimicrob. Chemother. 62:635– 637. http://dx.doi.org/10.1093/jac/dkn216. Ganesan LT, Manavathu EK, Cutright JL, Alangaden GJ, Chandrasekar PH. 2004. In-vitro activity of nikkomycin Z alone and in combination with polyenes, triazoles or echinocandins against Aspergillus fumigatus. Clin. Microbiol. Infect. 10:961–966. http://dx.doi.org/10.1111/j.1469 -0691.2004.00996.x. Shubitz LF, Roy ME, Nix DE, Galgiani JN. 2013. Efficacy of nikkomycin Z for respiratory coccidioidomycosis in naturally infected dogs. Med. Mycol. 51:747–754. http://dx.doi.org/10.3109/13693786.2013.770610. Horiuchi H. 2009. Functional diversity of chitin synthases of Aspergillus nidulans in hyphal growth, conidiophore development and septum formation. Med. Mycol. 47(Suppl 1):S47–S52. http://dx.doi.org/10.1080 /13693780802213332. Wang Q, Liu H, Szaniszlo PJ. 2002. Compensatory expression of five chitin synthase genes, a response to stress stimuli, in Wangiella (Exophiala) dermatitidis, a melanized fungal pathogen of humans. Microbiology 148(Pt 9):2811–2817. Liu H, Kauffman S, Becker JM, Szaniszlo PJ. 2004. Wangiella (Exophiala) dermatitidis WdChs5p, a class V chitin synthase, is essential for sustained cell growth at temperature of infection. Eukaryot. Cell 3:40 –51. http://dx.doi.org/10.1128/EC.3.1.40-51.2004. Weber I, Assmann D, Thines E, Steinberg G. 2006. Polar localizing class V myosin chitin synthases are essential during early plant infection in the plant pathogenic fungus Ustilago maydis. Plant Cell 18:225–242. http://dx .doi.org/10.1105/tpc.105.037341. Treitschke S, Doehlemann G, Schuster M, Steinberg G. 2010. The myosin motor domain of fungal chitin synthase V is dispensable for vesicle motility but required for virulence of the maize pathogen Ustilago maydis. Plant Cell 22:2476 –2494. http://dx.doi.org/10.1105/tpc.110.075028. Horiuchi H, Fujiwara M, Yamashita S, Ohta A, Takagi M. 1999. Proliferation of intrahyphal hyphae caused by disruption of CsmA, which encodes a class V chitin synthase with a myosin motor-like domain in Aspergillus nidulans. J. Bacteriol. 181:3721–3729. Cui Z, Wang Y, Lei N, Wang K, Zhu T. 2013. Botrytis cinerea chitin synthase BcChsVI is required for normal growth and pathogenicity. Curr. Genet. 59:119 –128. http://dx.doi.org/10.1007/s00294-013-0393-y. Jiménez-Ortigosa C, Aimanianda V, Muszkieta L, Mouyna I, Alsteens D, Pire S, Beau R, Krappmann S, Beauvais A, Dufrêne YF, Roncero C, Latgé JP. 2012. Chitin synthases with a myosin motor-like domain control the resistance of Aspergillus fumigatus to echinocandins. Antimicrob. Agents Chemother. 56:6121– 6131. http://dx.doi.org/10.1128/AAC.00752-12. Tsuizaki M, Takeshita N, Ohta A, Horiuchi H. 2009. Myosin motor-like domain of the class VI chitin synthase CsmB is essential to its functions in Aspergillus nidulans. Biosci. Biotechnol. Biochem. 73:1163–1167. http: //dx.doi.org/10.1271/bbb.90074.

Antimicrobial Agents and Chemotherapy

Downloaded from http://aac.asm.org/ on July 5, 2014 by GEORGE MASON UNIV

35.

using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402– 408. http://dx.doi.org/10.1006/meth.2001.1262. Kapteyn JC, Hoyer LL, Hecht JE, Muller WH, Andel A, Verkleij AJ. 2000. The cell wall architecture of Candida albicans wild-type cells and cell wall-defective mutants. Mol. Microbiol. 35:601– 611. http://dx.doi.org/10 .1046/j.1365-2958.2000.01729.x. Kahn JN, Hsu MJ, Racine F, Giacobbe R, Motyl M. 2006. Caspofungin susceptibility in Aspergillus and non-Aspergillus molds: inhibition of glucan synthase and reduction of beta-D-1,3 glucan levels in culture. Antimicrob. Agents Chemother. 50:2214 –2216. http://dx.doi.org/10.1128/AAC .01610-05. Fernández-Arenas E, Cabezon V, Bermejo C, Arroyo J, Nombela C, Diez-Orejas R, Gil C. 2007. Integrated proteomics and genomics strategies bring new insight into Candida albicans response upon macrophage interaction. Mol. Cell. Proteomics 6:460 – 478. http://dx.doi.org/10.1074 /mcp.M600210-MCP200. Moncunill G, Aponte JJ, Nhabomba AJ, Dobaño C. 2013. Performance of multiplex commercial kits to quantify cytokine and chemokine responses in culture supernatants from Plasmodium falciparum stimulations. PLoS One 8:e52587. http://dx.doi.org/10.1371/journal.pone.0052587. Ohm RA, Feau N, Henrissat B, Schoch CL, Horwitz BA, Barry KW, Condon BJ, Copeland AC, Dhillon B, Glaser F, Hesse CN, Kosti I, LaButti K, Lindquist EA, Lucas S, Salamov AA, Bradshaw RE, Ciuffetti L, Hamelin RC, Kema GH, Lawrence C, Scott JA, Spatafora JW, Turgeon BG, de Wit PJ, Zhong S, Goodwin SB, Grigoriev IV. 2012. Diverse lifestyles and strategies of plant pathogenesis encoded in the genomes of eighteen Dothideomycetes fungi. PLoS Pathog. 8:e1003037. http://dx.doi.org/10.1371/journal.ppat.1003037. Latgé JP. 2007. The cell wall: a carbohydrate armour for the fungal cell. Mol. Microbiol. 66:279 –290. http://dx.doi.org/10.1111/j.1365-2958.2007 .05872.x. Wiemann P, Sieber CM, von Bargen KW, Studt L, Niehaus M, Espino JJ, Huss K, Michielse CB, Albermann S, Wagner D, Bergner SV, Connolly LR, Fischer A, Reuter G, Kleigrowne K, Bald T, Wingfield BD, Ophir R, Freeman S, Hippler M, Smith KM, Brown DW, Proctor RH, Munsterkotter M, Freitag M, Humpf HU, Guldener U, Tudzynski B. 2013. Deciphering the cryptic genome: genome-wide analyses of the rice pathogen Fusarium fujikuroi reveal complex regulation of secondary metabolism and novel metabolites. PLoS Pathog. 9:e1003475. http://dx.doi .org/10.1371/journal.ppat.1003475. Martín-Urdíroz M, Roncero MI, González-Reyes JA, Ruiz-Roldán C. 2008. ChsVb, a class VII chitin synthase involved in septation, is critical for pathogenicity in Fusarium oxysporum. Eukaryot. Cell 7:112–121. http://dx .doi.org/10.1128/EC.00347-07. Bowman JC, Hicks PS, Kurtz MB, Rosen H, Schmatz DM, Liberator PA, Douglas CM. 2002. The antifungal echinocandin caspofungin acetate kills growing cells of Aspergillus fumigatus in vitro. Antimicrob. Agents Chemother. 46:3001–3012. http://dx.doi.org/10.1128/AAC.46.9.3001-3012 .2002. Frank U, Greiner M, Engels I, Daschner FD. 2004. Effects of caspofungin (MK-0991) and anidulafungin (LY303366) on phagocytosis, oxidative burst and killing of Candida albicans by human phagocytes. Eur. J. Clin. Microbiol. Infect. Dis. 23:729 –731. http://dx.doi.org/10.1007/s10096-004 -1171-z. Tullio V, Mandras N, Scalas D, Allizond V, Banche G, Roana J, Greco D, Castagno F, Cuffini AM, Carlone NA. 2010. Synergy of caspofungin with human polymorphonuclear granulocytes for killing Candida albicans. Antimicrob. Agents Chemother. 54:3964 –3966. http://dx.doi.org/10 .1128/AAC.01780-09. Hohl TM, Feldmesser M, Perlin DS, Pamer EG. 2008. Caspofungin modulates inflammatory responses to Aspergillus fumigatus through stage-specific effects on fungal ␤-glucan exposure. J. Infect. Dis. 198:176 – 185. http://dx.doi.org/10.1086/589304. Bush RK, Prochnau JJ. 2004. Alternaria-induced asthma. J. Allergy Clin. Immunol. 113:227–234. http://dx.doi.org/10.1016/j.jaci.2003.11.023. Pulimood TB, Corden JM, Bryden C, Sharples L, Nasser SM. 2007. Epidemic asthma and the role of the fungal mold Alternaria alternata. J. Allergy Clin. Immunol. 120:610 – 617. http://dx.doi.org/10.1016/j.jaci.2007.04 .045. Wheeler RT, Fink GR. 2006. A drug-sensitive genetic network masks